Project Summary/Abstract: Project 1 Nearly 53 million people live within 3 miles of a Superfund remediation site. Superfund sites contain a wide va- riety of pollutants, including organic chemicals, metals, carbonaceous material, and silica. Although each of these components is capable of damage to organisms, their potential to combine into unique hazardous agents has been understudied. Interestingly, when these agents are present together, particularly during thermal re- mediation, they combine to form particulate matter (PM) with chemisorbed free radicals that persist in the envi- ronment and biological systems. Over the prior funding cycle, we established that these environmentally per- sistent free radicals (EPFRs) represent underappreciated pollutant species, induce oxidative stress and dam- age, and negatively impact respiratory health. EPFRs are produced during thermal treatment (TT) of hazard- ous wastes and nearly 30% of all Superfund sites (excluding groundwater) are remediated by TT. EPFR con- centrations near Superfund sites range from 1x1018 - 4x1019 EPFRs/g (spins/g) of PM. This suggests a vast number of US residents are exposed to PM containing EPFRs. Short-term inhalational EPFR exposure elicited a Th17 inflammatory response similar to the severe asthma phenotype in humans. Mechanistically, we demonstrated a role for Th17 cells driven by the aryl hydrocarbon receptor (AhR) in this response. EPFRs also cause alterations in foreign compound metabolism. Not only is there a direct inhibition of P450-dependent activities, mediated by disruption of the complex between P450s and their redox partners, but EPFRs also induced P450s from the CYP1A family via AhR. Although there are unique features within the immunologic and metabolic aspects of the proposal, they share a common feature?AhR. Our hypothesis is that EPFRs modu- late AhR signaling leading to a pleiotropic response that alters both immunologic and P450 function resulting in poor respiratory health. Aim 1 will demonstrate that EPFR mediated activation of AhR in airway epithelial cells (AEC) induces Th17 responses. To test our hypothesis, we will employ mice in which AhR is selectively de- pleted from AECs. As part of this aim we will test the role of the tryptophan metabolite 6-formylindolo[3,2-b]car- bazole (FICZ) to activate AhR. Aim 2 will define the mechanism of EPFR inhibition of P450 function, focusing on P450 interactions with their redox partners NADPH-cytochrome P450 reductase (CPR) and cytochrome b5. These studies will show how inhibition of P450 function affects disposition of FICZ and ultimately Th17-medi- ated asthma. This will be accomplished by kinetic and biophysical examination of the effect of EPFRs on com- plex formation. Aim 3 will demonstrate an epidemiologic link between EPFR exposure and poor respiratory health in children. Measurements of glutathione sulfonamide, a biomarker of pulmonary neutrophilia and asso- ciated with Th17 mediated asthma, will be used to relate EPFR exposure and respiratory health. Responding to SRP Mandate 1 and 2, Project 1 will demonstrate that EPFR exposure impacts child respiratory health and provide mechanism(s) by which EPFRs contribute to disease.